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To assess the reliability and validity of age-specific versions of the Fibrodysplasia Ossificans Progressiva Physical Function Questionnaire (FOP-PFQ), developed to measure the impact of FOP on physical function and activities of daily living.FOP-PFQ development included a literature review, two iterative phases of qualitative work involving individuals with FOP, and clinical expert review. The analysis used pooled FOP-PFQ data from an FOP natural history study (NCT02322255), a patient registry (NCT02745158), and phase II trials (NCT02190747; NCT02279095; NCT02979769). Item-level and factor analysis informed item retention and determined factor structure. Reliability was evaluated using Cronbach's alpha and intraclass correlation coefficients. Convergent validity was assessed by comparing scores with age, the Cumulative Analogue Joint Involvement Scale (CAJIS), the Patient-Reported Outcomes Measurement Information System Global Health Scale (PROMIS), and heterotopic ossification (HO) volume. Known-groups validity assessment used age, CAJIS, and HO volume.Factor analysis confirmed a two-factor solution: Mobility and Upper Extremity. Results reflected high internal consistency and were supportive of test-retest reliability; correlation coefficients0.90 demonstrated FOP-PFQ scores were stable over a one- to three-week period. The majority of scores were moderately (r = 0.30-0.50) to highly (r ≥ 0.50) correlated with CAJIS and HO volume, supporting convergent validity. With the exception of some age-based and functional groups, FOP-PFQ scores were significantly worse in groups with more severe disease, demonstrating known-groups validity.The FOP-PFQ was demonstrated to be a reliable, valid measure that may be responsive to change in individuals with FOP, although some results were inconclusive for pediatric versions.

Purpose:To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing.Experimental Design:Pan-cancer MSI detection using Guardant360 was analytically validated according to established guidelines and clinically validated using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated.Results:cfDNA MSI evaluation was shown to have high specificity, precision, and sensitivity, with a limit of detection of 0.1% tumor content. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit.Conclusions:cfDNA-based MSI detection using Guardant360 is highly concordant with tissue-based testing, enabling highly accurate detection of MSI status concurrent with comprehensive genomic profiling and expanding access to immunotherapy for patients with advanced cancer for whom current testing practices are inadequate.See related commentary by Wang and Ajani, p. 6887

180 Background: ctDNA testing is a non-invasive means to identify genomic alterations in patients with aPC, including BRCA1/2 and other HRR gene alterations, which are targetable by PARP inhibitors (PARPi). However, classes of HRR inactivating mutations, such as copy number loss and large genomic rearrangements (LGRs) are challenging to detect by ctDNA. Here we describe a CLIA-validated plasma-based ctDNA test that identifies copy number loss and LGRs, in addition to SNV and Indels, in HRR genes. We report the landscape of HRR deficiency (HRD) in a clinical population of > 2,000 patients with aPC. Methods: 2932 samples from patients with aPC previously tested as part of clinical care using an 83-gene cfDNA NGS assay (Guardant360) within an 11-month period were reanalyzed for presence of HRD. HRD alterations were defined as copy number loss, LGR (i.e. intergenic or intragenic gene rearrangements) and deleterious SNV/Indel in 7 clinically focused HRR genes ( ATM, BRCA1, BRCA2, CDK12, CHEK2, PALB2, RAD51D). Clinical information, when available, was obtained from test requisition forms. Results: The median age of the analyzed cohort was 73 years (range: 31-100); when known, the majority of patients were tested at disease progression (81%, 2242/2750). In total, an inactivating BRCA1/2 variant was was identified in 26% (605/2932) of reanalyzed patients, comprising BRCA1/2 loss of heterozygosity (LOH) (13.4% of patients), deleterious SNV or Indels (5.5%), homozygous deletions (3.3%) and LGRs (1.1%). The prevalence of patients with any inactivating HRR alteration increased to 38.5% with the expanded 7 gene set. Overall, prevalence of loss-of-function mutations (SNV/Indel, homozygous deletion or LGR) in BRCA2 was 4.5X frequent than in BRCA1 and comparable to previously described prostate cohorts (8.4% and 1.8% compared to 8.6% and 1.1% in tissue, respectively). Conclusions: Genomic profiling by ctDNA identified an HRR alteration landscape comparable to previously characterized tissue cohorts, with enrichment in BRCA2 over BRCA1 inactivating mutations and frequent BRCA1/2 LOH. Patients with aPC may benefit from PARPi eligibility determination using rapid and reliable ctDNA assessment, particularly at progression. Future studies should assess PARPi outcomes for this population.

Background RNA viruses such as HCV and HIV mutate at extremely high rates, and as a result, they exist in infected hosts as populations of genetically related variants. Recent advances in sequencing technologies make possible to identify such populations at great depth. In particular, these technologies provide new opportunities for inference of relatedness between viral samples, identification of transmission clusters and sources of infection, which are crucial tasks for viral outbreaks investigations. Results We present (i) an evolutionary simulation algorithm Viral Outbreak InferenCE (VOICE) inferring genetic relatedness, (ii) an algorithm MinDistB detecting possible transmission using minimal distances between intra-host viral populations and sizes of their relative borders, and (iii) a non-parametric recursive clustering algorithm Relatedness Depth (ReD) analyzing clusters’ structure to infer possible transmissions and their directions. All proposed algorithms were validated using real sequencing data from HCV outbreaks. Conclusions All algorithms are applicable to the analysis of outbreaks of highly heterogeneous RNA viruses. Our experimental validation shows that they can successfully identify genetic relatedness between viral populations, as well as infer transmission clusters and outbreak sources.

Rapidly evolving RNA viruses continuously produce minority haplotypes that can become dominant if they are drug-resistant or can better evade the immune system. Therefore, early detection and identification of minority viral haplotypes may help to promptly adjust the patient's treatment plan preventing potential disease complications. Minority haplotypes can be identified using next-generation sequencing (NGS), but sequencing noise hinders accurate identification. The elimination of sequencing noise is a non-trivial task that still remains open. Here we propose CliqueSNV based on extracting pairs of statistically linked mutations from noisy reads. This effectively reduces sequencing noise and enables identifying minority haplotypes with the frequency below the sequencing error rate. We comparatively assess the performance of CliqueSNV using an in vitro mixture of nine haplotypes that were derived from the mutation profile of an existing HIV patient. We show that CliqueSNV can accurately assemble viral haplotypes with frequencies as low as 0.1% and maintains consistent performance across short and long bases sequencing platforms.

Hepatitis C virus (HCV) usually establishes chronic infection, which is often asymptomatic at the early stages of disease. Unfortunately, no diagnostic criteria that can distinguish between recent and chronic HCV infections are available. Error-prone replication of HCV causes each patient to host a heterogeneous population of genetically related HCV variants. Therefore, it is usually supposed that intra-host HCV heterogeneity gradually increases over the course of infection. However, due to the complex nature of the structural development of HCV populations inside hosts being influenced by selective sweeps and negative selection during chronic infection [3][2], the accuracy of simple metrics for the assessment of genetic heterogeneity is insufficient for HCV infection staging.

Background: Late stage therapy selection is advancing beyond the detection of classical driver alterations (SNV, Indel, CNV, Fusion) to the use of aggregate biomarkers comprising combinations of SNVs, Indels, and fusions, that reflect the underlying mutational process, indicate the cellular dysfunction, and suggest the neoantigen signature. GuardantOMNI (OMNI), is a highly sensitive 500-gene cfDNA sequencing test designed to detect primary somatic mutations as well as aggregate biomarkers including TMB and DDR pathway defects. The newest version of OMNI introduces MSI biomarker detection, which is an indication for the first FDA-approved pan-cancer therapy. Here we present the first analytical validation study of OMNI MSI-H detection. Methods: Probabilistic models for MSI detection were designed to interrogate instability across >1300 loci, differentiating PCR and sequencing induced artifacts from true biological signals using molecular barcoding as implemented in the Guardant digital sequencing platform. Cut-offs were selected based on the analysis of the training healthy donor cohort and locked prior to validation. Analytical performance was assessed using pre-characterized cell lines, cancer-free donor-samples, and clinical patient samples. Qualitative and quantitative orthogonal confirmation was provided by next-generation sequencing (NGS) and published information. Results: 350 samples were processed for this study, using both 5ng and 30ng cfDNA input levels, and all samples passed sequencing QC metrics established prior to testing. Cell line-based dilution studies performed in triplicate spanning 5ng and 30ng input range as well as in silico simulation demonstrated 95% limit of detection (LoD) at 0.1% Analysis of 121 healthy donor and 16 MSS advanced cancer patient samples identified no false positives (FP). In 240 patient samples with orthogonal confirmation of MSI status from tissue or panel NGS, GuardantOMNI demonstrated 100% analytic sensitivity (3/3) and 100% analytic specificity (237/237) with a PPV of 1 and and NPV 1. Retrospective analysis of 2000+ clinical samples detected MSI high in 16% of colorectal cancers, 4% of prostate cancers, and 0.6% of non-small cell lung cancers. Conclusions: Here we have demonstrated the performance of MSI-H detection with GuardantOMNI (OMNI), which has the potential to accelerate clinical trial enrollment, research and discovery with a single, non-invasive blood sample. Citation Format: Jing Zhao, Alexander Artyomenko, Carlo Artieri, John Latham, Stephen R. Fairclough, Catalin Barbacioru, Elena Helman, John Strickler, Darya Chudova, Richard Lanman, AmirAli Talasaz. Analytical validation of MSI High detection with GuardantOMNI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1675.

Introduction: Cell-free circulating tumor DNA (cfDNA) is utilized for genomic alteration detection to inform therapy selection and is an emerging noninvasive disease monitoring tool. Nucleic acid-based therapies (NATs) directly or indirectly manipulate DNA or mRNA transcript levels for therapeutic effect. cfDNA is in routine clinical use and NATs are in an expanding number of clinical trials, yet little is known about potential interactions of these technologies including the need for cfDNA next-generation sequencing (NGS) to recognize this source of exogenous DNA. Methods: A 62-year-old male with weight loss and abdominal pain was found to have a tumor in the head of the pancreas and a metastatic liver lesion. Fine-needle aspirate of the tumor confirmed mucinous adenocarcinoma of the pancreas but sample was insufficient for molecular testing. cfDNA NGS was performed (Guardant Health, CA) to identify genomic alterations (e.g., BRCA, EGFR alterations, ERBB2 amplification). Results: cfDNA NGS revealed dramatic over-representation (41% of all reads) of TP53-derived sequences with atypical insert sizes and distribution, read start/stop positions, and post-clipping read lengths. Atypical sequences comprised only TP53 coding sequence, without introns. Alignment and reconstruction assembled the atypical reads into a 6003 bp circular construct with the complete TP53 coding sequence inserted into a synthetic expression vector. Consultation with the treating physician revealed that patient progressed on FOLFIRINOX and was subsequently enrolled on a clinical trial utilizing a liposome-encapsulated TP53 expression vector with gemcitabine and nab-paclitaxel. His first experimental treatment was four days prior to cfDNA sample collection. Importantly, over-representation of this synthetic construct obscured native TP53 sequences, resulting in a 47% increase in predicted assay limit of detection despite standard performance metrics being within normal ranges. The atypical features of the expression vector reads make it possible to automate detection and quantification, even at significantly lower concentration than described here. The expression vector bears no human genome homology, allowing read identification via mapping. TP53 intron “deletions” were detected by a novel deletion detection algorithm. Finally, insert length and read start-stop distributions are markedly dissimilar between native and synthetic sequences Conclusions: NATs generate unique and previously unappreciated interference with cfDNA sequencing which can be identified and filtered using automatable and generalizable bioinformatics techniques. cfDNA sequencing identified an NAT construct, four days after administration, demonstrating that the ability to identify and quantify NATs may enable noninvasive assessment of NAT delivery efficacy and/or pharmacologic persistence. Citation Format: Marcin Sikora, Alexander Artyomenko, James Strauss, Richard B. Lanman, Victoria M. Raymond, Justin Odegaard. Cell-free DNA sequencing demonstrates persistence of nucleic acid-based therapy—opportunities for detection and monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3662.

Pacific Biosciences (PacBio) sequencing is providing thousands of reads with the length up to 10,000 bases. In most cases this length is enough to cover entire region of interest however this technology has high (≈15%) error rate. We propose a method for viral haplotype reconstruction using long SMRT reads. The proposed method based on correlation between SNVs and maximum likelihood for frequency estimation. When applied to PacBio reads from an Influenza A Virus (IAV) sample with ten variants, our method was able to reconstruct the nine most frequent.

Motivation: Next-generation sequencing (NGS) allows for analyzing a large number of viral sequences from infected patients, providing an opportunity to implement large-scale molecular surveillance of viral diseases. However, despite improvements in technology, traditional protocols for NGS of large numbers of samples are still highly cost and labor intensive. One of the possible cost-effective alternatives is combinatorial pooling. Although a number of pooling strategies for consensus sequencing of DNA samples and detection of SNPs have been proposed, these strategies cannot be applied to sequencing of highly heterogeneous viral populations. Results: We developed a cost-effective and reliable protocol for sequencing of viral samples, that combines NGS using barcoding and combinatorial pooling and a computational framework including algorithms for optimal virus-specific pools design and deconvolution of individual samples from sequenced pools. Evaluation of the framework on experimental and simulated data for hepatitis C virus showed that it substantially reduces the sequencing costs and allows deconvolution of viral populations with a high accuracy. Availability and implementation: The source code and experimental data sets are available at http://alan.cs.gsu.edu/NGS/?q=content/pooling Contact: kki8@cdc.gov, yek0@cdc.gov Supplementary information: Supplementary data are available at Bioinformatics online.

We present a deterministic version of our novel Monte-Carlo Regression based method MCReg [1] for transcriptome quantification from RNA-Seq reads. Experiments on simulated and real datasets demonstrate better transcriptome frequency estimation accuracy compared to that of the existing tools which tend to skew the estimated frequency toward super-transcripts.